BCIDT represent an important advancement in therapy for RA. However, there remains a proportion of patients who do not improve despite therapy. Although BCIDT have been shown to be highly efficient and safe for the treatment of RA patients, clinical complications have been reported for a subgroup of the treated patients. These include events associated with the first infusion (transient hyo- or hyper tension, pruritus and rash) and an increased incidence of infections (Cohen S. B. et al., Rituximab for rheumatoid arthritis refractory to anti-tumor necrosis factor therapy: Results of a multicenter, randomized, double blind, placebo controlled, phase III trial evaluating primary efficacy and safety at twenty-four weeks. Arthritis Rheum 2006, 54:2793-2806). These drugs are expensive and have the potential of serious toxicity. Therefore, it would be ideal to predict the patients who will respond, so that the use of these drugs can be targeted. Methods of predicting BCIDT response known in the art are based on observational, habitual or demographic variables (age, sex, disease activity score (DAS28) or health assessment questionnaire (HAQ) scores (Hydrich et al. Rheum 2006: 1558-1565). There is a need is for improved methods using personalised biological parameters. BCIDT have been implied for B- and T-cell, and auto-antibody—associated autoimmune diseases (AAID) such as multiple sclerosis (Hauser S. L. et al. B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N. Engl. J. med. 2008, 358; 676-688), Grave's disease (Fassi L. et al. Treatment of Grave's disease with rituximab specifically reduces the production of thyroid stimulating antibodies. Clin. Immunol. 2009, 130:352-358), Wegener's disease, Pemphigus Vulgaris (Ahmed A. R. et al., Treatment of pemphigus vulgaris with rituximab and intravenoud immuneglobulin. N. Engl. J. Med. 2006, 54:2970-2982; Mouquet et al., B-cell depletion immunotherapy in pemphigus: effects on cellular and humoral immune responses. J. Invest. Derm. 2008, 128:2859-2869), systemic lupus erythematosus (Gunnarsson I. et al., Histopathologic and clinical outcome of rituximab treatment in patients with cyclophosphamide-resistant proliferative lupus nephritis. Arth. Rheum 2007, 56:1263-1272; Guzman R. A. et al., Rituximab in refractory systemic lupus erythemathosus. Lupus 2005, 14: 221 (OP18)), Sjogren's syndrome (Guzman R. A. et al, Rituximab in primary sjogren syndrome. J. Clin. Rheumatol. 2006, 12: 164 (s52)), some forms of vasculitis, some types of inflammatory muscle disease (Guzman R. A. et al., B cell depletion in poly-dermatomyositis. 6th international Congress on Autoimmunity. Porto Portugal 2008 (URL kenes[dot]com[slash]autoimmunity)), systemic sclerosis, type I diabetes and immune and thrombotic thrombocytopenic purpura (Stasi R. Et al., Rituximab chimeric anti-CD20-monoclonal antibody treatment for adults with chronic idiotpathic thrombocytopaenic purpura. Blood 2001, 98: 952-957). It is desirable to predict whether a patient will respond to BCIDT.
The invention relates to a method for prognosticating the clinical response of a patient to treatment with a soluble BCID or TCID agent, said method comprising the steps of                a. Obtaining at least two samples from said patient wherein a first sample has not been exposed to a soluble BCID or TCID agent and wherein at least a second sample has been exposed to a soluble BCID or TCID agent        b. Determining the level of an IFN-I type response in said at least two samples,        c. Comparing the level of the IFN-I type response in said first sample with the level of the IFN-I type response in said at least second sample and        d. Prognosticating said clinical response from said comparison.        
The method may also be performed by obtaining a sample that has not been exposed to soluble BCIDT or TCIDT before the start of therapy and prognosticate the clinical response by comparing the level of IFN response gene expression to a cut-off point
The term “patient” refers to any subject (preferably human) afflicted with a disease likely to benefit from BCIDT, in particular a B-cell-related disease. B cells are the precursors of antibody-producing cells (plasma cells). In the process of undergoing activation and maturation into memory B cells and plasma cells they are very efficient antigen presenting cells (APCs) to T cells of soluble antigens that are bound specifically by the B cell antigen receptor (surface immunolglobuline). B cell ontogeny is characterized by a series of changing surface phenotypes. One of these is the CD20 surface marker (a 33-37 kDa membrane associated phosphoprotein) expressed during intermediate stages of development, which is lost during terminal differentiation to the immunoglobulin producing plasma cell. The exclusivity and high specificity of B-cell molecules like CD20 makes these types of proteins attractive pharmaceutical targets. Specifically beneficial features of CD20 are that free CD20 is not present in the circulation, CD20 does not modulate its own expression, and CD20 is not shed or internalised after antibody binding. Moreover, no endogeneous CD20-like molecules are known that interfere with its function (Press et al., Monoclonal antibody 1F5 (anti-CD20) serotherapy of B-cell lymphomas. Blood 1987, 69:584-591). Diseases wherein B-cells directly contribute to pathogenesis and/or indirectly influence disease via changes in T cell function can be efficiently treated with BCIDT. B cell targeting via anti-CD20, e.g. rituximab (an anti-CD20 antibody), rapidly depletes peripheral blood CD20 positive B cells via complement-mediated and antibody dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis and inhibition of cell growth (Maloney D. G. et al., Rituximab: Mechanism of action and resistance. Semin. Oncol. 2002, 29:2-9). B-cell levels usually reach a minimum by 1 month and repopulation generally starts by 6 months. Rituximab also downregulates CD40 ligand, CD40 and CD80, resulting in cganges to T cell function (Tokunaga M. Et al., Downregulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab. Rheumatology 2005, 44:176-182). It is not yet certain which of the possible mechanisms of action is most important in vivo. Interestingly, marked variability between individual responses have been observed, with a portion of patients failing to achieve a clinical response and others who reach a clinician remission for over 2 years.
In a preferred embodiment, said patient suffers from a disease selected from the group consisting of a B- or T-cell related disease, and an auto-antibody-associated autoimmune diseases (RAID). These diseases are likely to benefit from BCIDT.
Preferred diseases are selected from the group consisting of multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, some forms of vasculitis, some types of inflammatory muscle disease, systemic sclerosis, type I diabetes and immune and thrombotic thrombocytopenic purpura, and transplant rejection or graft-versus-host disease, malignancy, a pulmonary disorder, an intestinal disorder, a cardiac disorder, a spondyloarthropathy, a metabolic disorder, anemia, pain, a hepatic disorder, and a skin disorder. In one embodiment, the autoimmune disorder is selected from the group consisting of rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, and nephrotic syndrome. In another embodiment, said B- and T-cell, and auto-antibody-associated autoimmune diseases are selected from the group consisting of inflammatory bone disorders, bone resorption disease, periodontal disease. In still another embodiment, said B- and T-cell, and auto-antibody-associated autoimmune diseases are selected from the group consisting of Behcet's disease, ankylosing spondylitis, asthma, chronic obstructive pulmonary disorder (COPD), idiopathic pulmonary fibrosis (IPF), restenosis, diabetes, anemia, pain, a Crohn's disease-related disorder, juvenile rheumatoid arthritis (JRA), psoriatic arthritis, and chronic plaque psoriasis.
In one embodiment of the invention, the B- and T-cell, and auto-antibody-associated autoimmune disease is Crohn's disease. In another embodiment, the disease is ulcerative colitis. In still another embodiment, the disease is psoriasis. In still another embodiment, the disease is psoriasis in combination with psoriatic arthritis (PsA).
In another preferred embodiment, said B- and T-cell, and auto-antibody-associated autoimmune disease comprises a disease which is likely to benefit from BCIDT. Preferably, said disease comprises type I diabetes.
In a preferred embodiment, said patient is an individual suffering from at risk or suffering from “Rheumatoid Arthritis (RA)” With the term an individual suffering from at risk or suffering from Rheumatoid Arthritis (RA) is meant an individual who is diagnosed with RA or is suspected by a doctor of suffering from RA or of developing the symptoms of RA within 10 years. Predominant symptoms of RA comprise pain, stiffness, and swelling of peripheral joints. The clinical manifestation of the disorder is very variable, ranging from mild, self-limiting arthritis to rapidly progressive multi-system inflammation with profound morbidity and mortality (Lee & Weinblatt 2001; Sweeney & Firestein 2004). RA symptoms may also comprise joint damage, which typically occurs early in the course of rheumatoid arthritis; 30 percent of patients have radiographic evidence of bony erosions at the time of diagnosis, and this proportion increases to 60 percent by two years (van der Heijde 1995; Br J. Rheumatol., vol. 34 Suppl 2, pp. 74-78). Typically, RA is a polyarthritis, which involves many joints (six or more), although for example in the early stages of the disease, only one or a few joints might be afflicted. Virtually all peripheral joints can be affected by the disease; however, the most commonly involved joints are those of the hands, feet and knees (Smolen et al, 1995; Arthritis Rheum., vol. 38, no, 1, pp. 38-43). In addition, RA can affect the spine, and atlanto-axial joint involvement is common in longer-standing disease, and constitutes a directly joint-related cause of mortality. Extra-articular involvement is another hallmark of RA, and this can range from rheumatoid nodules to life-threatening vasculitis (Smolen & Steiner 2003; Nat. Rev. Drug Discov., vol. 2, no. 6, pp. 473-488). RA can be classified using history, physical examination, laboratory and radiographic findings and this is usually performed according to criteria as described in Arnett F C, et al.: Arthritis Rheum 31:315, 1988. Preferably, said individual has a DAS28 score of 3.2 or higher. More preferably, said DAS28 score is 4.6 or higher. Most preferably, said DAS28 score is 5.1 or higher.
Patients who are resistant to methotrexate (MTX), usually considered first-line therapy for the treatment of RA, and/or failed to respond to TNF-blockers, are a further preferred group of patients for whom the method of the invention can be particularly useful.
More generally, patients who already receive a basic treatment for their TNF-related disease, e.g. with or without MTX, azathioprine or leflunomide, are particularly good candidates for the test method of the invention.
With the term “clinical response” is meant the clinical result of BCIDT. Said clinical response can be a positive or a negative clinical response. With a positive clinical response is meant that the severity of symptoms or the number of symptoms is reduced as a result of BCIDT or TCIDT. Preferably, said clinical result is the result of a treatment with a soluble TNF antagonist. When the disease is RA, it is preferred that a positive clinical response comprises at least reduction of swelling of joints. Preferably, an assessment of a clinical response is based on standardized and preferably validated clinical response criteria such as provided by the guidelines of organisations such as the National Institute for Health and Clinical Excellence (NICE), EULAR and/or ACR. Preferred clinical response criteria comprise DAS, DAS28 or the EULAR criteria or a combination thereof. In a preferred embodiment determination of a clinical response is based on an assessment using the DAS28 criteria. An advantage of the DAS28 is that it very sensitive to small effects of a therapy. Therefore, the method is very accurate when using DAS28 criteria.
Preferably, a positive clinical response is defined as a reduction in DAS28 score of at least 1.2 compared to the score of said individual prior to treatment with a soluble TNF antagonist. More preferably, a clinical response is based on assessment using EULAR criteria and DAS28 criteria.
Even more preferably, clinical response criteria are combined with demographic data, other clinical information or information about relevant habits. Demographic data comprise gender and/or age. Clinical information may comprise any relevant clinical observation or data. Preferred clinical information comprises CRP, ESR, ACPA titre, IgM RF titre, disease duration and medication. Information about relevant habits may be any relevant information. Preferred information comprises information about smoking habits.
B lymphocyte dysregulation with the production of rheumatoid factor (RF) and other autoantibodies, formation of immune complexes and release of destructive mediators are known to contribute to RA pathogenesis (Mannik M. and Nardella F. A., IgG rheumatoid factors and self-association of these antibodies, Clin. Rheum. Dis. 1985, 11:551-572). Approximately 80% of the RA patients develop RF antibodies. It is thought that B cells that produce RF migrate into the synovium and activate T cells by presentation of an antigen bound to IgG via HLA-DR4, via uptake by surface bound RF (Edwards J. C. W. and Cambridge G., Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes. Rheumatology 2001, 40: 205-211). It was hypothesized that by eliminating this B cell antigen presentation to synovial T cells with anti-Cd20, T cell activation and T cell dependent synovial inflammation would decrease. In addition the ability of IgG RF B cells to self perpetuate, due to secretion of own antigen, provided rationale for the proposal that eradication of these cell clones may result in prolonged disease remission (Edwards J. C. W. et al., Do self-perpetuating B lymphocytes drive human auto-immune disease? Immunology 1999, 97:1868-1896).
The term “BCIDT” refers to molecules, such as proteins or small molecules, which can significantly reduce B cell function and/or number, and/or T cell function.
Preferably said BCIDT comprise anti-B cell antibodies, e.g. rituximab (Chimeric IgG1 Genentech/Biogen Approved 1997), Y90-Ibritumomab tiuxetan (Murine (90Y) NHL Biogen/IDEC Low ADCC Approved 2002), I131tositumomab (Murine (131I) NHL GSK Low CDC Approved 2003), Ofatumumab (Human IgG1 NHL/RA Genmab AC/GSK High CDC and ADCC Phase III trials), Ocrelizumab (Humanised IgG1 NHL/RA Genentech/Roche/Biogen Phase III trials), TRU-015 (SMIP # RA Trubion Pharma/Wyeth High ADCC Phase I/II Low CDC), Veltuzumab (Humanised NHL and ITP Immunomedics Phase I/II IgG1), AME-133v (Humanised IgG1 Relapsed NHL Applied Molecular High ADCC Phase I/II Evolution/Eli Lilly), PRO131921 (Humanised IgG1 CLL and NHL Genentech High CDC and ADCC Phase I/II (Version 114), GA10168 (Humanised CLL and NHL Glycart/Roche High PCD and ADCC Phase I/II), and anti-T cell antibodies e.g. Abatacept (recombinant fusion protein that selectively modulates CD80 and CD86-CD28 costimulatory signal required for full T cell activation), and alefacept (bivalent recombinant fusion protein consisting of a LFA-3 portion that binds CD2 receptors on T-cells, IgG1 portion of alefacept binds to Fc▭R receptor on natural killer cells to induce T-cell apoptosis).
Preferred therapies with soluble B and T-cell inhibitory or depleting molecules of the invention include, for example, rituximab, Y90-Ibritumomab tiuxetan, I131tositumomab, Ofatumumab, Ocrelizumab, and anti-T cell antibodies e.g. abatacept, and alefacept. More preferably, said soluble B and T-cell inhibitory or depleting molecule comprises rituximab.
With the term “sample” is meant any suitable sample comprising proteins or nucleotides. Preferred suitable samples include whole blood, saliva, faecal material, buccal smears, skin, and biopsies of specific organ tissues, such as muscle or nerve tissue and hair follicle, because these samples comprise relevant expression products. Preferably, said cell sample is a blood sample, because a blood sample is easy obtainable and comprises large amounts of relevant expression products.
With the term “IFN-I type response” is meant a response comprising the expression of an expression product of a gene involved in the IFN-I pathway. With the level of an IFN-I type response is meant the amount of expression product of any gene involved in the IFN-I response pathway.
An “expression product” of a gene is RNA produced from said genes or a protein produced from said RNA. The levels of the expression products may be determined separately for each different expression product or as a single measurement for more different expression products simultaneously. Preferably, the determination of the level of the expression products is performed for each different expression product separately, resulting in a separate measurement of the level of the expression product for each different expression product. This enables a more accurate comparison of expression levels of expression products with the expression levels of the same expression products in a control.
Determination of the level of the expression products according to methods of the invention may comprise the measurement of the amount of nucleic acids or of proteins. In a preferred embodiment of the invention, determination of the level of the expression products comprises determination of the amount of RNA, preferably mRNA. A level can be the absolute level or a relative level compared to the level of another mRNA. mRNA can be isolated from the samples by methods well known to those skilled in the art as described, e.g., in Ausubel at al., Current Protocols in Molecular Biology, Vol. 1; pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc. (1996). Methods for detecting the amount of mRNA are well known in the art and include, but are not limited to, northern blotting, reverse transcription PCR, real time quantitative PCR and other hybridization methods. The amount of mRNA is preferably determined by contacting the mRNAs with at least one sequence-specific oligonucleotide which hybridises to said mRNA. In a preferred embodiment said mRNA is determined with two sequence-specific oligonucleotides which hybridise to different sections of said mRNA. The sequence-specific oligonucleotides are preferably of sufficient length to specifically hybridize only to the RNA or to a cDNA prepared from said mRNA. As used herein, the term “oligonucleotide” refers to a single-stranded nucleic acid. Generally the sequence-specific oligonucleotides will be at least 15 to 20 nucleotides in length, although in some cases longer probes of at least 20 to 25 nucleotides will be desirable. Said sequence-specific oligonucleotides may also comprise non-specific nucleic acids. Such non-specific nucleic acids can be used for structural purposes, for example as an anchor to immobilise the oligonucleotides. The sequence-specific oligonucleotide can be labelled with one or more labelling moieties to permit detection of the hybridized probe/target polynucleotide complexes. Labelling moieties can include compositions that can be detected by spectroscopic, biochemical, photochemical, bioelectronic, immunochemical, and electrical optical or chemical means. Examples of labelling moieties include, but are not limited to, radioisotopes, e.g., 32P, 33P, 35S, chemiluminescent compounds, labelled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, linked enzymes, mass spectrometry tags, and magnetic labels. Oligonucleotide arrays for mRNA or expression monitoring can be prepared and used according to techniques which are well known to those skilled in the art as described, e.g., in Lockhart et al., Nature Biotechnology, Vol. 14, pp. 1675-1680 (1996); McGall et al., Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 13555-13460 (1996); and U.S. Pat. No. 6,040,138.
A preferred method for determining the amount of mRNA involves hybridization of labelled mRNA to an ordered array of sequence-specific oligonucleotides. Such a method allows the simultaneously determination of the mRNA amounts. The sequence-specific oligonucleotides utilized in this hybridization method typically are bound to a solid support. Examples of solid supports include, but are not limited to, membranes, filters, slides, paper, nylon, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, polymers, polyvinyl chloride dishes, etc.
According to a preferred embodiment of the invention the determining the level(s) of the expression products is performed by measuring the amount of protein. The term “protein” as used herein may be used synonymously with the term “polypeptide” or may refer to, in addition, a complex of two or more polypeptides which may be linked by bonds other than peptide bonds, for example, such polypeptides making up the protein may be linked by disulfide bonds. The term “protein” may also comprehend a family of polypeptides having identical amino acid sequences but different post-translational modifications, particularly as may be added when such proteins are expressed in eukaryotic hosts. These proteins can be either in their native form or they may be immunologically detectable fragments of the proteins resulting, for example, from proteolytic breakdown. By “immunologically detectable” is meant that the protein fragments contain an epitope which is specifically recognized by e.g. mass spectrometry or antibody reagents as described below. Proteins levels can be determined by methods known to the skilled person, comprising but not limited to: mass spectrometry, Western blotting, immunoassays, protein expression assay, protein microarray etc.
A preferred embodiment of the invention provides a protein microarray (Templin at al. 2004; Comb. Chem. High Throughput Screen., vol. 7, no. 3, pp. 223-229) for simultaneous binding and quantification of the at least two biomarker proteins according to the invention. The protein microarray consists of molecules (capture agents) bound to a defined spot position on a support material. The array is then exposed to a complex protein sample. Capture agents such as antibodies are able to bind the protein of interest from the biological sample. The binding of the specific analyte proteins to the individual spots can then be monitored by quantifying the signal generated by each spot (MacBeath 2002; Nat. Genet, vol. 32 Suppl, pp. 526-532; Zhu & Snyder 2003; Curr. Opin. Chem. Biol., vol. 7, no. 1, pp. 55-63). Protein microarrays can be classified into two major categories according to their applications. These are defined as protein expression microarrays, and protein function microarrays (Kodadek 2001; Chem. Biol., vol. 8, no. 2, pp. 105-115). Protein expression microarrays mainly serve as an analytic tool, and can be used to detect and quantify proteins, antigen or antibodies in a biological fluid or sample. Protein function microarrays on the other hand can be used to study protein-protein, enzyme-substrate and small molecule-protein interactions (Huang 2003; Front Biosci., vol. 8, p. d559-d576). Protein microarrays also come in many structural forms. These include two-dimensional microarrays constructed on a planar surface, and three-dimensional microarrays which use a Flow-through support.
Types of protein microarray set-ups: reverse phase arrays (RPAs) and forward phase arrays (FPAs) (Liotta et al. 2003; Cancer Cell, vol. 3, no. 4, pp. 317-325). In RPAs a small amount of a tissue or cell sample is immobilized on each array spot, such that an array is composed of different patient samples or cellular lysates. In the RPA format, each array is incubated with one detection protein (e.g., antibody), and a single analyte endpoint is measured and directly compared across multiple samples. In FPAs capture agents, usually an antibody or antigen, are immobilized onto the surface and act as a capture molecule. Each spot contains one type of immobilized antibody or capture protein. Each array is incubated with one test sample, and multiple analytes are measured at once.
One of the most common forms of FPAs is an antibody microarray. Antibody microarrays can be produced in two forms, either by a sandwich assay or by direct labelling approach. The sandwich assay approach utilizes two different antibodies that recognize two different epitopes on the target protein. One antibody is immobilized on a solid support and captures its target molecule from the biological sample. Using the appropriate detection system, the labelled second antibody detects the bound targets. The main advantage of the sandwich assay is its high specificity and sensitivity (Templin, Stoll, Bachmann, & Joos 2004; Comb. Chem. High Throughput. Screen., vol. 7, no. 3, pp. 223-229). High sensitivity is achieved by a dramatic reduction of background yielding a high signal-to noise ratio. In addition, only minimal amounts of labelled detection antibodies are applied in contrast to the direct labelling approach were a huge amount of labelled proteins are present in a sample. The sandwich immunoassay format can also be easily amenable to the field of microarray technology, and such immunoassays can be applied to the protein microarray format to quantify proteins in conditioned media and/or patient sera (Huang et at 2001; Clin. Chem. Lab Med., vol. 39, no. 3, pp. 209-214; Schweitzer et at 2002; Nat Biotechnol., vol. 20, no. 4, pp. 359-365).
In the direct labelling approach, all proteins in a sample are labelled with a fluorophore. Labelled proteins that bind to the protein microarray such as to an antibody microarray are then directly detected by fluorescence. An adaptation of the direct labelling approach is described by Haab and co-workers (Haab, Dunham, & Brown 2001; Genome Biol., vol. 2, no. 2, p). In this approach, proteins from two different biological samples are labelled with either Cy3 or Cy5 fluorophores. These two labelled samples are then equally mixed together and applied to an antibody microarray. This approach, for example, allows comparisons to be made between diseased and healthy, or treated and untreated samples. Direct labelling has several advantages, one of which is that the direct labelling method only requires one specific antibody to perform an assay.
Miniaturized and multiplexed immunoassays may also used to screen a biological sample for the presence or absence of proteins such as antibodies (Joos et al. 2000; Electrophoresis, vol. 21, no. 13, pp. 2641-2650; Robinson et al. 2002; Nat. Med., vol. 8, no. 3, pp. 295-301).
In a preferred embodiment of the invention, the detection or capture agents such as the antibodies are immobilized on a solid support, such as for example on a polystyrene surface. In another preferred embodiment, the detection or capture agents are spotted or immobilized in duplicate, triplicate or quadruplicate onto the bottom of one well of a 96 well plate.
In a method according to the invention, a first sample is tested that has not been exposed to a soluble BCIDT and/or TCIDT. The level of an IFN-I type response of this sample is determined as a control sample. The level of an IFN-I type response of this sample is compared to the level of an IFN-I type response of a second sample from said individual. It is preferred that the first sample and the second sample are of the same tissue.
It is required that the second sample has been exposed to BCIDT and/or TCIDT. A sample from an individual who had received said soluble BCID and/or TCID treatment can be used.
Moreover, for response prediction a cell sample from an untreated individual can be used wherein said cell sample has been contacted with a soluble BCID or TCID agent in vitro. It is preferred that all samples have been provided with the same soluble BCID or TCID agent.
Accordingly, it is possible to perform a method according to the invention wherein said at least a second sample has been contacted with soluble BCID or TCID agents in vitro, whereas said first of said samples has been provided with a soluble BCID or TCID agent. This method is also preferred. An advantage thereof is that an in vitro culture with a soluble BCID or TCID agent is technically easier to perform. When a cell sample is used from an individual who had been treated with a soluble BCID or TCID agent, it is preferred to use a sample that is collected at some time after said individual had been exposed to said soluble BCID or TCID agent to allow said soluble BCID or TCID agent to interact with said sample and to allow INF type I genes to respond to said soluble BCID or TCID agent. Preferably, a cell sample is used which is collected between 1 and 4 months after the first exposure to said soluble BCID or TCID agent. More preferably, a cell sample which is collected between at 1-3 months after exposure is used, because at said time points, differences between good and poor responders are greater. Expression of genes involved in the type I IFN pathway reaches its peak around 3 months after starting a treatment with a soluble BCID or TCID agent. It is preferred that at least two cell samples are collected between 1 and 4 months after exposure to a soluble BCID or TCID agent is used, because more samples from different time points increases the accuracy of the method. Most preferably, a cell sample collected at 1, 2, 3 and 4 months after exposure to soluble BCID or TCID agent is used.
When using a cell sample from an individual who did not receive an a treatment with said soluble soluble BCID or TCID agent, said cell sample is preferably exposed to a soluble BCID or TCID agent under in vitro conditions. For in-vitro culturing conditions, said cell sample is preferably a blood sample. Preferably, said conditions comprise culturing cells. Culturing procedures for different cell types are well known in the art and a skilled person will be able to select a suitable procedure for the selected cell types.
A method of the invention is also suited to prognosticate the clinical response of an individual to said soluble BCID or TCID agent, prior to starting a treatment of said individual. To this end, the first sample is tested that has not been exposed to a soluble BCID or TCID agent. An advantage thereof is that such method can be used to determine the prospect of a positive clinical response in individuals before the start of BCIDT and/or TCIDT. The level of an IFN-I type response of this sample from the said individual is determined.
Moreover, a method of the invention is also suited to prognosticate the clinical response of an individual to said soluble BCID or TCID agent, prior to starting a treatment of said individual if at least two samples are cultured in vitro, in the presence and absence of a soluble BCID or TCID agent. It is understood that if the method is performed using in-vitro exposure of a sample, said first and said second samples may have been collected as a single sample which is split into a first and a second sample. An advantage thereof is that such method can be used to determine the prospect of a positive clinical response in individuals before the start of BCIDT or TCIDT.
It is preferred that said soluble BCID or TCID agent is allowed to interact with said cells and to allow genes involved in the type I IFN pathway to respond to the soluble BCID or TCID agent before measuring expression levels of said genes. Preferably, a preferred moment for measuring said expression levels is when the response of said genes is at its peak. A skilled person will be able to establish the most suitable moment to do this by culturing a cell sample taken from an individual prior to therapy with a soluble BCID or TCID agent. Preferably, culturing conditions comprise culturing in the presence of a soluble BCID or TCID agent for 24 to 48 hours. Preferably, said samples comprise blood cells. Preferably, said sample comprises whole blood.
In the present invention we show that some RA patients display a significant difference in the peripheral blood gene expression level of IFN type I response genes in association with a significant difference in the magnitude of the treatment-induced expression of type I IFN-response genes.
In a method according to the invention, an individual has a increased prospect of a positive clinical response to a treatment with a soluble BCID or TCID agent if expression levels of the expression products of IFN response genes of a said treatment are low prior to the start of treatment or are higher compared to the levels of the same expression products of said first sample. A increased prospect of a positive clinical response to a treatment with soluble soluble BCID or TCID agent is thus by a low level of expression products of IFN response genes in the first sample taken prior to the start of therapy, and/or increased if the in-vitro by a soluble BCID or TCID agent induced expression of IFN response genes is increased compared to levels of the same products of said first sample.
However, in another group of treated RA patients an increased level of expression of IFN-response genes is observed in the first sample taken prior of the strat of rituximab treatment. Especially RA patients that showed a poor clinical response to treatment showed an increase in IFN-response gene expression levels before the start of treatment and a decreased or no difference in IFN-response gene expression levels compared to that of sample one of the same individual after treatment with rituximab.
The present invention confirms the existence of both regulatory routes since some RA patients actively increase and others decrease their type I IFN-response genes upon rituximab.
In summary, this study shows that there is a large variation in the change of IFN-response gene expression levels before and after therapy with soluble BCID or TCID agents between RA patients. Interestingly, increased levels of gene products of type I IFN response genes or treatment-induced downregulation or an expression of IFN response genes which is not significantly different from that in the first sample take prior to the start of in-vivo or in-vitro administration of rituximab is associated with a poor clinical response to rituximab treatment. Monitoring of the IFN-response genes Mx1, ISG15, BAFF, DARC, OAS1, LGALS3BP, Mx2, OAS2 and SERPING1 before and after the start of rituximab is useful for early determination of clinical response to treatment. Consequently, the opposite effect of that observed with a good clinical response to rituximab. Hence, low levels of type I IFN response gene activity and an in-vivo or in-vitro induced upregulation of IFN response genes is associated with a good clinical response to a soluble BCID or TCID agent.
An expression level is classified as increased at baseline, i.e. prior to the start of therapy with a soluble BCID or TCID agent when said expression level of said expression product of said first sample is statistically significantly increased in said individual compared to the level of the same expression product found in a sample of a healthy control individuals. The term “significantly” or “statistically significant” refers to statistical significance and generally means a two standard deviation (SD) above normal, or higher, or below, or lower concentration of the expression product. In preferred embodiments, said difference is classified as statistically significant if the expression level is at least a 20 percent increased compared to expression level of the same expression product in control individuals. Preferably, the increase or decrease is at least 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or 250 percent. Most preferably, said increase or decrease is at least 100 percent.
An expression level is also classified as different when said expression level of said expression product of said second sample is statistically significantly increased or decreased in said individual compared to the level of the same expression product found in said first sample. The term “significantly” or “statistically significant” refers to statistical significance and generally means a two standard deviation (SD) above normal, or higher, or below, or lower concentration of the expression product. In preferred embodiments, said difference is classified as statistically significant if the expression level is at least a 20 percent increased or decreased compared to expression level of the same expression product in control individuals. Preferably, the increase or decrease is at least 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or 250 percent. Most preferably, said increase or decrease is at least 100 percent.
More preferred is a method, wherein said IFN-I type response level is determined by determining in said first sample or at least two samples the level of an expression product of at least one gene of Table 2. An advantage thereof is that these genes are more predictive. More preferably, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or 34 genes of Table 2 are used, because the inclusion of more genes of this table improves the accuracy of the method.
More preferred is a method, wherein one level of said IFN-I type response is determined by determining the level of an expression product of at least ISG15, Mx1, OAS1, LGALS3BP, RSAD2, IFI44L, MX2, OAS2, BAFF, DARC and/or SERPING1. An advantage thereof is that these genes have a good predictive power.
In a preferred embodiment of the invention, said IFN-I type response is determined by determining the level of an expression product of at least OAS1, ISG15 and Mx1. An advantage thereof is that these use of these genes results in a good predictive power.
In another preferred embodiment of the invention, said IFN-I type response is determined by determining the level of an expression product of at least OAS1, ISG15 and Mx1. These genes have a good predictive power.
Even more preferred is an embodiment, wherein said IFN-I type response is determined by further determining the level of an expression product of at least Mx1, OAS1, and ISG15. An advantage thereof is that the combined use leads to an improved predictive power.
Another preferred embodiment is a method wherein said IFN-I type response is determined by determining the level of an expression product of at least Mx1, LGALS3BP, RSAD2, OAS1, ISG15, and IFI44L. More preferably, said IFN-I type response is determined by determining the level of an expression product of at least Mx1, OAS1, ISG14, LGALS3BP, Mx2, OAS2 and SERPING1.
More preferably said IFN-I type response is determined by determining the level of an expression product of at least the 15 validation genes listed in Table 2 and 3, and BAFF and DARC. This further improves the predictive power of the method. Most preferred is a method wherein at least the 34 genes listed (Van der Pouw Kraan C. T. M. at al., Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients. Annals of rheumatic Dis. 2007, 66:1008-1014).
In another aspect, the invention relates to a method for prognosticating a clinical response of a patient to a treatment with a soluble BCD or TCID agent, said method comprising determining the level of the expression products of the genes listed in Table 2 and 3, and BAFF and DARC in the said first sample prior to the start of therapy with soluble BCID or TCID agent, or at least two samples of said individual, wherein a first of said samples has not been exposed to a soluble BCID or TCID agent and wherein at least a second of said samples has been exposed to a soluble BCID or TCID agent prior to determining said level, said method further comprising comparing said levels and prognosticating said clinical response from said comparison.
More preferred is a method wherein said at least one gene comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 genes of Table 2 and 3, and BAFF and DARC.
More preferred is a method wherein said at least two samples comprise cell samples. An advantage thereof is that cells samples comprise nucleic acids, which can advantageously be used for determining said levels of an IFN-I type response, said level of expression product of said genes and/or said at least one gene listed in Table 2 and 3, and BAFF and DARC.
More preferred is a method wherein second sample is of an individual between 1 and 4 months after the first exposure of said individual to said soluble BCID or TCID agent. An advantage thereof is that within this period, said IFN-I type response level or said level of the expression products of Table 3 differs significantly compared to said first sample.
In another aspect the invention relates to a method for treatment of a patient with a soluble BCID or TCID agent, comprising determining a prognosis for a clinical response to a treatment with said soluble BCID or TCID agent, further comprising treating said individual with said soluble soluble BCID or TCID agent, if said individual has been prognosticated as a good responder.
In another aspect, the invention relates to use of a soluble BCD or TCID agent for the preparation of a medicament for the treatment of a patient, wherein prior to said treatment a prognosis for a clinical response to said soluble BCID or TCID agent was determined with any of the methods described above.
In another aspect, the invention relates to an improved pharmacodynamic marker (PD marker) for evaluating a pharmacological effect of a treatment with a soluble BCID or TCID agent. Good PD markers are needed to improve the prediction of the efficacy and safety of a treatment with a soluble BCID or TCID agent at the individual patient level. These quantitative PD markers should reflect features of drug exposure and drug response with respect to modulation of the molecular target, the cognate biochemical pathways and/or downstream biological effects. The availability of quantitative PD markers provides a rational basis for decision making during e.g. treatment optimization. A PD marker currently described for rituximab is peripheral blood B cell levels. Various reasons for inadequate depletion have been proposed, including genetic polymorphisms of the FcRyIIIa gene (Anolik J. H. et al., The relationship of FCyRIIIa genotype to the degree of B cell depletion by rituximab in the treatment for systemic lupus eruthematosus. Arthritis Rheum 2003, 48:455-459) or defective complement. Other PD markers include levels of RF and autoantibodies against citrullinated are described to be downregulated in patients treated with soluble BCID or TCID agent who show a clinical response (Cambridge G. Et al., Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis. Arthritis Rheum 2003, 48: 2146-2154). Synovial biopsy studies suggested that the clinical response was associated with degree of B cell depletion in the synovial per se, but not in synovial plasma cell numbers and immunoglobuline production (Thurlings R. M. et al. Synovial tissue response to rituximab: mechanism of action and identification of biomerkers of response. Ann. Rheum. Dis. 2008, 67: 917-925). However, neither fully explains the response status. Moreover, most of the described PD markers are assessed by using mean levels of patient groups while most of these markers are not affected in each individual patient.
The invention provides a method for evaluating a pharmacological effect of a treatment of a patient with a soluble BCID or TCID agent said method comprising determining the level of an expression product of at least one gene of Table S2 in at least two samples of said individual, wherein a first of said samples has not been exposed to a soluble BCID or TCID agent and wherein at least a second of said samples has been exposed to said soluble BCID or TCID agent prior to determining said level. The method is preferably used to determine whether moment of renewed therapy and dose of a soluble BCID or TCID agent which a patient receives is well timed and sufficiently high to achieve an effect or a clinical response. Whether a clinical response can be achieved depends also on other factors. The method can also be used to determine whether the dose of a soluble BCID or TCID agent which a patient receives is not too high and might therefore cause side effects. With the term “pharmacological effect” is meant a biochemical or physiological effect of a soluble BCID or TCID agent. Preferably, such pharmacological effect is specific for a treatment with a soluble BCID or TCID agent. Preferably, such pharmacological effect reflects the relationship between an effective dose and the clinical response. Preferably, said effective dose is the dose as measured in the blood level. With the term “evaluating” is meant that results of a pharmacological effect is determined and used for decision making steps regarding further treatment. Preferably, evaluating comprises evaluating the dose, the efficacy and/or the safety of said soluble BCID or TCID agent. Preferably, the expression products of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, genes of Table 2 and 3 and BAFF and DARC are used in the method. Other terms used are explained above. Preferably, the expression products of said genes comprises the genes, wherein the level of said expression product is higher than 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.4, 2.6 or 3.0, or lower than 0.68, 0.67, 0.66, 0.65, 0.64, 0.62 or 0.60 (see column “fold change”, Table 2 and 3 and BAFF and DARC). Values equal or lower than T3 values and in “fold change” and reaching baseline expression values in Table 2 and 3 and BAFF and increased DARC are indicative of renewed administration and eventually increased dose of a soluble BCID or TCID agent. Values higher than 1 in “fold change” and resembling 13 values as listed in Table 2 and 3 and BAFF and DARC are indicative of an prolonged renewal of treatment. Upregulation of a gene having a “fold change” higher than 1 as listed in table 3 is indicative of an effective dose of a soluble BCID or TCID agent. In another preferred embodiment, said at least one gene comprises preferably genes and gene products that are responsive to type I IFN. See table 2 for further details on the mentioned genes. Preferred is a method wherein at least said second of said samples has been exposed to a soluble BCID or TCID agent prior to determining said level
Another preferred is a method wherein at least said second of said samples has been exposed to a soluble BCID or TCID agent prior to determining said level.
An advantage of this method is that said level of at least one gene of Table 2 and 3 and BAFF and DARC reflects a good clinical response to a therapy with a soluble BCID or TCID agent. Therefore, said response reflects drug activity and can be used to monitor drug efficacy at the individual patient level. Drug efficacy is the ability of a drug to produce the desired therapeutic effect.
In another aspect, the invention relates to a method for treatment of a patient with a soluble BCID or TCID agent, wherein the dose of said soluble BCID or TCID agent treatment is based on results obtained by a method for evaluating a pharmacological effect of a treatment of a patient with a soluble BCID or TCID agent said method comprising determining the level of a pharmacogenomic response of at least one gene of Table 2 and 3 and BAFF and DARC in at least two samples of said individual, wherein a first of said samples has not been exposed to a a soluble BCID or TCID agent and wherein at least a second of said samples has been exposed to a soluble BCID or TCID agent prior to determining said level. The term “based on” means that results of said method are taken into account in establishing the dose of said a soluble BCID or TCID agent most suited for the individual patient. Preferred is a method wherein a patient is treated with a soluble BCID or TCID agent and wherein said method for evaluating a pharmacological response is based on results obtained by a method for evaluating a pharmacological effect wherein said at least a second of said samples has been exposed to a soluble BCID or TCID agent prior to determining said level.
In another aspect, the invention relates to use of a soluble BCID or TCID agent for the preparation of a medicament for the treatment of an patient, wherein said wherein said treatment is evaluated based on a method for evaluating a pharmacological effect of a treatment of a patient with a soluble BCID or TCID agent said method comprising determining the level of a pharmacogenomic response of at least one gene of table 2 and BAFF and DARC in at least two samples of said individual, wherein a first of said samples has not been exposed to a soluble BCID or TCID agent and wherein at least a second of said samples has been exposed to said a soluble BCD or TCID agent prior to determining said level. Another preferred embodiment is the use of a soluble BCID or TCID agent for the preparation of a medicament for the treatment of a patient and wherein said method for evaluating a pharmacological response is based on results obtained by a method for evaluating a pharmacological effect wherein said at least a second of said samples has been exposed to a soluble BCID or TCID agent prior to determining said level.
In another aspect, the invention relates to a kit suitable for use in the above method, comprising up to 34 reagents, sequence specific oligonucleotides and/or capture agents for detecting up to 34 of the gene products listed (Van der Pouw Kraan C. T. M. et al., Rheumatoid arthritis subtypes identified by genomic profiling of peripheral blood cells: assignment of a type I interferon signature in a subpopulation of patients. Annals of rheumatic Dis. 2007, 66:1008-1014).
and of the genes listed in Table 2 and 3.
In another aspect, the invention relates to a kit suitable for use in the above method, comprising up to table 2 and 3 and BAFF and DARC.